Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies.
Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen.
CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) Tcells or bispecific Tcell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with Tcell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.
View details for DOI 10.1016/j.cell.2020.08.022
View details for PubMedID 32961131
Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation.
The majority of tumor-infiltrating T cells exhibit a terminally exhausted phenotype, marked by a loss of self-renewal capacity. How repetitive antigenic stimulation impairs T cell self-renewal remains poorly defined. Here, we show that persistent antigenic stimulation impaired ADP-coupled oxidative phosphorylation. The resultant bioenergetic compromise blocked proliferation by limiting nucleotide triphosphate synthesis. Inhibition of mitochondrial oxidative phosphorylation in activated T cells was sufficient to suppress proliferation and upregulate genes linked to T cell exhaustion. Conversely, prevention of mitochondrial oxidative stress during chronic T cell stimulation allowed sustained T cell proliferation and induced genes associated with stem-like progenitor T cells. As a result, antioxidant treatment enhanced the anti-tumor efficacy of chronically stimulated T cells. These data reveal that loss of ATP production through oxidative phosphorylation limits T cell proliferation and effector function during chronic antigenic stimulation. Furthermore, treatments that maintain redox balance promote T cell self-renewal and enhance anti-tumor immunity.
View details for DOI 10.1038/s41590-020-0725-2
View details for PubMedID 32661364
Human B Cell Clonal Expansion and Convergent Antibody Responses to SARS-CoV-2.
Cell host & microbe
The Xist lncRNA mediates X chromosome inactivation (XCI)1,2. Here we show that Spen, an Xist-binding repressor protein essential for XCI3-9, binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen inactivation activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing10-11. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.
View details for DOI 10.7554/eLife.54508
View details for PubMedID 32379046
Rapid and reversible suppression of ALT by DAXX in osteosarcoma cells.
2019; 9 (1): 4544
B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV.
View details for DOI 10.1016/j.chom.2020.09.002
View details for PubMedID 32941787
Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion.
2019; 37 (8): 925?36
Many tumors maintain chromosome-ends through a telomerase-independent, DNA-templated mechanism called alternative lengthening of telomeres (ALT). While ALT occurs in only a subset of tumors, it is strongly associated with mutations in the genes ATRX and DAXX, which encode components of an H3.3 histone chaperone complex. The role of ATRX and DAXX mutations in potentiating the mechanism of ALT remains incompletely understood. Here we characterize an osteosarcoma cell line, G292, with wild-type ATRX but a unique chromosome translocation resulting in loss of DAXX function. While ATRX and DAXX form a complex in G292, this complex fails to localize to nuclear PML bodies. We demonstrate that introduction of wild type DAXX suppresses the ALT phenotype and restores the localization of ATRX/DAXX to PML bodies. Using an inducible system, we show that ALT-associated PML bodies are disrupted rapidly following DAXX induction and that ALT is again restored following withdrawal of DAXX.
View details for DOI 10.1038/s41598-019-41058-8
View details for PubMedID 30872698
Tracking the immune response with single-cell genomics.
Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here, we assess the performance of a massively parallel droplet-based method for mapping transposase-accessible chromatin in single cells using sequencing (scATAC-seq). We apply scATAC-seq to obtain chromatin profiles of more than 200,000 single cells in human blood and basal cell carcinoma. In blood, application of scATAC-seq enables marker-free identification of cell type-specific cis- and trans-regulatory elements, mapping of disease-associated enhancer activity and reconstruction of trajectories of cellular differentiation. In basal cell carcinoma, application of scATAC-seq reveals regulatory networks in malignant, stromal and immune cells in the tumor microenvironment. Analysis of scATAC-seq profiles from serial tumor biopsies before and after programmed cell death protein 1 blockade identifies chromatin regulators of therapy-responsive T cell subsets and reveals a shared regulatory program that governs intratumoral CD8+ T cell exhaustion and CD4+ T follicular helper cell development. We anticipate that scATAC-seq will enable the unbiased discovery of gene regulatory factors across diverse biological systems.
View details for DOI 10.1038/s41587-019-0206-z
View details for PubMedID 31375813
Clonal replacement of tumor-specific T cells following PD-1 blockade.
The immune system is composed of a diverse array of cell types, each with a specialized role in orchestrating the immune response to pathogens or cancer. Even within a single cell 'type,' individual cells can access a wide spectrum of differentiation and activation states, which reflect the physiological response of each cell to the tissue environment and immune stimuli. Thus, the cellular diversity of the immune system is inherently quite complex and understanding this complexity has greatly benefited from technologies that measure immune responses at single-cell resolution, in addition to the systems-level response as a whole. In this Commentary, we focus on recent work at the interface of immunology and single-cell genomics and highlight advances in technologies and their application to immune cells. In particular, we highlight recent single-cell genomic profiling studies of T cells, since somatic rearrangements in the T cell receptor (TCR) loci enable the tracking of clonal T cell responses through space and time. Finally, we discuss opportunities for future use of these technologies in understanding vaccination and the basis for effective vaccine-induced immunity.
View details for DOI 10.1016/j.vaccine.2019.11.035
View details for PubMedID 31859202
Promoter of lncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element.
2018; 173 (6): 1398
Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of patients with cancer1. However, whether the T cell response to checkpoint blockade relies on reinvigoration of pre-existing tumor-infiltrating lymphocytes or on recruitment of novel T cells remains unclear2-4. Here we performed paired single-cell RNA and T cell receptor sequencing on 79,046 cells from site-matched tumors from patients with basal or squamous cell carcinoma before and after anti-PD-1 therapy. Tracking T cell receptor clones and transcriptional phenotypes revealed coupling of tumor recognition, clonal expansion and T cell dysfunction marked by clonal expansion of CD8+CD39+ T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, the expansion of T cell clones did not derive from pre-existing tumor-infiltrating T lymphocytes; instead, the expanded clones consisted of novel clonotypes that had not previously been observed in the same tumor. Clonal replacement of T cells was preferentially observed in exhausted CD8+ T cells and evident in patients with basal or squamous cell carcinoma. These results demonstrate that pre-existing tumor-specific T cells may have limited reinvigoration capacity, and that the T cell response to checkpoint blockade derives from a distinct repertoire of T cell clones that may have just recently entered the tumor.
View details for DOI 10.1038/s41591-019-0522-3
View details for PubMedID 31359002
ATAC Primer Tool for targeted analysis of accessible chromatin
2018; 15 (5): 304?5
Pembrolizumab for advanced basal cell carcinoma: an investigator-initiated, proof-of-concept study.
Journal of the American Academy of Dermatology
Noncoding mutations in cancer genomes are frequentbut challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth invivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.
View details for PubMedID 29731168